Artificial insemination of equine mares has been of importance for many years. Often this has been accomplished surgically. In routine instances where lower dosages of sperm have not been required, it has been accomplished without surgery by artificial insemination, however this has used relatively high numbers of sperm. For routine artificial insemination of the mare 250-500×106 progressively motile sperm (pms) inseminated every other day to a mare in estrus, is usually recommended to achieve maximum fertility. Unfortunately, when inseminating mares with semen from a highly fertile stallion, fertility has decreased as the number of motile sperm has been reduced. Under ideal conditions, a mare has been successfully inseminated with as few as 100×106 pms without reducing fertility, however even this number poses challenges. Insemination with low numbers of sperm is necessary when using sorted sexed semen and frozen thawed semen of limited quantity.
Preselecting the sex of offspring in an equine animal is, of course, of interest. Sex preselection following artificial insemination (AI) with low numbers of separate, enriched populations of X- and Y-chromosome bearing sperm that have been separated on the basis of DNA content is currently possible in other species, however, equine species have in some regards proven more difficult. While birth of progeny of the desired sex following intrauterine insemination of cattle and sheep has validated the sexing technology, until the present invention, it has not been practically applied to equines.
To achieve sex preselection involves separating the X- from the Y-chromosome bearing sperm followed by use for artificial insemination (AI) or for in vitro fertilization (IVF) and subsequent embryo transfer. Current high speed flow cytometry enables researchers to sort >1000 live-sexed sperm/second. Sorting alone, however, is not enough. In order to make semen sexing a practical technique for a commercial equine AI program, a lesser number of motile sperm is required for an insemination dose. In the mare when using fresh semen for AI, a typical insemination dose would contain between 250-500×106 motile sperm. The current sorting rate of ˜1000 live sperm/second, it would take almost six days to sort one insemination dose! Therefore, a lesser number of motile sperm is required to practically achieve reasonable fertility. Once that is achieved, enhanced fertility with non-surgical insemination of mares with sexed semen is also viewed as practically necessary.
As mentioned, sex preselection involves the use of DNA content and separation of sperm into X- and Y-chromosome bearing populations. Using current high speed flow cytometry enables researchers to sort up to 1000 live sperm/sec of the desired sex with 90% accuracy, which provides adequate numbers of sperm in many species other than equines in a reasonable amount of time. For example, this new technology for sperm sexing has made it a practical technique for artificial insemination in cattle. Since it is practical to sort only a low number of spermatozoa and still maintain sperm viability, one aspect of this invention addresses enhanced pregnancy rates following insemination of 25×106 non sorted, progressively motile spermatozoa (pms). This is about one-tenth of what was previously considered optimal for routine operations. Other aspects address even lower numbers. These aspects can have significant economic consequences when one considers its application to celebrated trophy animals such as horses and the like.
As mentioned, one of the fundamental challenges that efforts at sorting X and Y equine sperm has faced is the large numbers of sperm involved. In natural insemination equine sperm are produced by nearly the billion; in artificial insemination less, but still significantly large numbers of equine sperm are usually used. For instance, equine artificial insemination techniques routinely use two hundred and fifty million to five hundred million sperm. Thus a significant number of sperm have been presumed necessary in an equine artificial insemination environment.
As the invention relates to sex selected artificial insemination, many methods have been attempted to achieve the separation of X- and Y-chromosome bearing sperm in other animals. None of these, however, have dealt with aspects peculiar to or specific sorting of equine sperm cells. General sorting methods have ranged from magnetic techniques such as appears disclosed in U.S. Pat. No. 4,276,139 to columnar techniques as appears disclosed in U.S. Pat. No. 5,514,537 to gravimetric techniques as discussed in U.S. Pat. No. 3,894,529, reissue Pat. No. 32,350, U.S. Pat. Nos. 4,092,229, 4,067,965, and 4,155,831. Electrical properties have also been attempted as shown in U.S. Pat. No. 4,083,957 as well as a combination of electrical and gravimetric properties as discussed in U.S. Pat. Nos. 4,225,405, 4,698,142, and 4,749,458. Motility efforts have also been attempted as shown in U.S. Pat. Nos. 4,009,260 and 4,339,434. Chemical techniques such as those shown in U.S. Pat. Nos. 4,511,661 and 4,999,283 (involving monoclonal antibodies) and U.S. Pat. Nos. 5,021,244, 5,346,990, 5,439,362, and 5,660,997 (involving membrane proteins), and U.S. Pat. Nos. 3,687,803, 4,191,749, 4,448,767, and 4,680,258 (involving antibodies) as well as the addition of serum components as shown in U.S. Pat. No. 4,085,205. While each of these techniques has been presented as if to be highly efficient, in fact at present none of those techniques yield the desired level of sex preselection and none have shown success at the artificial insemination level with equine sperm. Regardless of the separation technique eventually used, however, the competing combinations of the high numbers of equine sperm naturally present and the approach of separating X- and Y-chromosome bearing sperm has made it desirable to develop an ability to achieve equine insemination with lower numbers of sperm.
The quantitative technique used to achieve the separation of X- and Y-chromosome bearing sperm for artificial insemination (of any species) has been that involving the technique of flow cytometry. This technique appeared possible as a result of advances and discoveries involving the differential dye absorption of X- and Y-chromosome bearing sperm. This was discussed early in U.S. Pat. No. 4,362,246 and significantly expanded upon through the techniques disclosed by Lawrence Johnson in U.S. Pat. No. 5,135,759. The Johnson technique of utilizing flow cytometry to separate X- and Y-chromosome bearing sperm has been so significant an advancement that it has for the first time made the commercial separation of such sperm feasible. Further, separation has been significantly enhanced through the utilization of high speed flow cytometers such as the MoFlo® flow cytometer produced by Cytomation, Inc. and discussed in a variety of other patents including U.S. Pat. Nos. 5,150,313, 5,602,039, 5,602,349, and 5,643,796 as well as international PCT patent publication WO 96/12171. While the utilization of Cytomation's MoFlo® cytometers has permitted great increases in speed, and while these speed increases are particularly relevant given the high number of equine sperm often used, certain problems have still remained. In spite of the almost ten-fold advances in speed possible by the MoFlo® flow cytometer, shorter and shorter sorting times have been desired for several reasons. First, it has been discovered that as a practical matter, the equine sperm are time-critical cells. They lose their effectiveness the longer they remain unused. Second, the collection, sorting, and insemination timings has made speed an item of high commercial importance. Thus, the time critical nature of the equine sperm cells and of the process has made speed an essential element in achieving high efficacy and success rates in artificial insemination.
In spite of some successes in sorting and then artificially inseminating animals of other species, the effort with equines has proven particularly elusive. As relevant to the present invention, equine applications may have been particularly challenging either because the equine conception process and/or the equine sperms cells themselves are more delicate than those of other species—especially bovines. For this reason, it may even be that those skilled in the art have not viewed techniques or systems developed for other species as applicable to equines. In some instances almost identical procedures from a non-equine species do not provide the same type of result for equines. This may have fostered separation in the research efforts and in the techniques and substances developed.
Other problems also exist ranging from the practical to the theoretical. On the practical side, it has been desired to achieve equine artificial insemination in a manner that can be done in the field rather than a laboratory environment. Thus, for commercial production and success in the field, improvements which might only represent an increase in efficiency or practicality may still be significant. Related to the practical aspect, is the aspect of the delicateness and sensitivity of the entire process. In this regard, it has been desired to simplify the process and make it as procedurally robust as possible so that operator error or skill can play an ever decreasing role. This goal has also combined to make insemination with lower dosages even more desirable.
In addition to the delicateness of the process, it has always been known that the sperm in general are extremely delicate cells. While this factor at first glance seems like it might be considered easily understood, in fact, the full extent of the cells' sensitivities have not yet been fully explored. Furthermore equine sperm appear particularly sensitive. In contrast to bovine sperm, they are in many ways more delicate from the perspective of successful artificial insemination. Different sensitivities arise and thus there has to some degree been a perception that the systems, techniques, and substances used in other animals (such as bovines) may not always be adaptable to equines. This has in fact proven to be true.
In the context of flow cytometry in general, most sorted cells or particles have often been physically able to withstand a variety of abuses. This is not the case for equine sperm cells. In fact, as the present invention discloses, the processing through normal flow cytometer techniques may, in fact, be unacceptable for cytometric sorting of equine sperm cells in certain applications. The sensitivities range from dilution problems and the flow cytometer's inherent need to isolate and distinguish each cell individually as well as the pressure and other stress which typical flow cytometry has (prior to the present invention) imposed upon the equine cells it was sorting. This may also represent a unique factor for equine sperm cells because it appears that even though the equine sperm cell may appear to pass through the flow cytometer and be sorted with no visually discernable side-effects, in fact, the cells themselves may have been stressed to the point that they perform less than optimally in the insemination process. Thus, an interplay of factors seems involved and has raised unusual problems from the perspective of equine sperm cell sorting and ultimate use for equine artificial insemination.
Another problem which has remained—in spite of the great advances achieved through the Johnson patent and related technology—is the fact that prior to the present invention it has been extremely difficult to achieve lower dosage insemination with sexed equine sperm, regardless of the separation technology used. While historically, some achievement of low dose insemination has occurred, it has appeared to be more in a theoretical or laboratory environment rather than in environments which are likely to be experienced in or applicable to a commercial application. It has also occurred through surgical techniques. In this regard, the desire has not been merely to achieve low dose insemination but even to achieve non-surgical insemination in a field environment. To achieve low dose insemination with pregnancy success rates which are comparable to existing unsexed, high dosage artificial insemination efforts is thus quite significant. The advances achieved by the present inventors in both sexed, unsexed, and low dose artificial insemination represent significant advances which may, for the first time, make commercial applications feasible to equids.
Another problem which has been faced by those in the industry—again, in spite of the great advances by the Johnson patent and related technology—is the fact that the problem itself, namely, equine artificial insemination with a high success rate is one of a statistical nature in which a multitude of factors seem to interplay. Thus, the solutions proposed may to some degree involve a combination of factors which, when thoroughly statistically studied, will be shown to be necessary either in isolation or in combination with other factors. Such a determination is further compounded by the fact that the results themselves vary by species and may be difficult to ascertain due to the fact that testing and statistical sampling on a large enough data base is not likely to be worth the effort at the initial stages. For these reasons the invention can also involve a combination of factors which may, individually or in combination, represent the appropriate solutions for a given application. This disclosure is thus to be considered broad enough so that the various combinations and permeations of the techniques disclosed may be achieved. Synergies may exist with other factors. Such factors may range from factors within the sorting, or perhaps, flow cytometer, steps to those in the collection as well as insemination steps. Thus, while there has been a long felt but unsatisfied need for high speed, low dose sexed equine insemination, and while certain of the implementing arts and elements have long been available, prior to the present invention the advances or perhaps combinations of advances had apparently been overlooked by those skilled in the art. It may even be that the proper combination of known elements simply was not realized. Perhaps to some degree those in the field may have failed to appreciate that the problem involved an interplay of factors as well as peculiar necessities for equine sperm cells involved in this field. Interestingly, as the listing of efforts later in this discussion shows, substantial attempts had been made but they apparently failed to understand the problem inherent in such an area as low dose, sexed insemination of equines and had perhaps assumed that because the natural service event involves perhaps a billion sperm, there may have been physical limitations to the achievement of artificial insemination with numbers which are as many as three orders of magnitude less in number. Thus, it may not be surprising that there was to some extent an actual teaching away from the technical direction in which the present inventors went. Perhaps the results may even be considered unexpected to a degree because they have shown that sexed, low dose equine artificial insemination—if done right—can be achieved with success rates comparable to those of unsexed, high dose equine artificial insemination. It might even be surprising to some that the techniques and advances of the present invention in fact combine to achieve the great results shown. While each technique might, in isolation, be viewed by some as unremarkable, in fact, the subtle changes or combination with other techniques appear to afford significant advances in the end result.
Thus, in one regard until the present invention the achievement of non-surgical practical equine artificial insemination low dose, sexed artificial insemination of equines has not been possible with levels of performance necessary or simplified procedures likely to be necessary to achieve commercial implementation. Beyond low dose, sexed insemination on a commercial level, however achieved, the present invention also discloses techniques which permit the achievement of improved performances and thus facilitates the end result desired, namely, low dose, sexed and unsexed non-surgical artificial insemination of equines on a commercial basis.